The Unit of Molecular Signal Transduction investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides are a small fraction of the cellular phospholipids, but play a critical role in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases and GTP-binding proteins, as well as membrane transporters including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis and metabolism. Our group focuses on one family of enzymes, the phosphatidylinositol 4-kinases (PI4Ks) that catalyze the first committed step in phosphoinositide synthesis. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases. The cellular effects of many hormones and neurotransmitters are initiated by receptor-activated hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) to generate two important second messengers, Ins(1,4,5)P3 (InsP3) and diacylglycerol (DAG). InsP3 rapidly binds to its intracellular receptors and releases Ca2+ from non-mitochondrial Ca2+ stores to increase cytoplasmic Ca2+, while DAG activates the various isoforms of PKC, both triggering specific cellular responses of the target cell. We have shown previously that the sustained formation of these messengers depends on the activity of the type III PI 4-kinases (PI4Ks). None of the two type-III PI4Ks are detectable by immunfluorescence in the plasma membrane, where PI(4)P must be generated for conversion to PI(4,5)P2. We have used small interfering RNA (siRNA) to down-regulate the levels of the four cellular PI4Ks and applied the pleckstrin-homology (PH) domains of the OSBP, FAPP1 and OSH2 proteins fused to GFP to monitor PI(4)P production in single COS-7 cells. The OSBP and FAPP1 PH domains could report on PI(4)P synthesis in the plasma membrane but only after recovery from a robust PLC activation evoked by the addition of the Ca2+ ionophore, ionomycin. In contrast, the OSH2 PH domain can detect PI(4)P in the plasma membrane without stimulation of PI turnover. The plasma membrane localization of PH-OSBP and PH-FAPP1 after recovery from Ca2+ stimulation was prevented by wortmannin, which inhibits both type-III PI4Ks, and by a concentration (10 ?M) of phenylarsine-oxide (PAO) that specifically inhibits the type-IIIalpha but not the type-IIIbeta enzyme. The localization of both PH domains was not affected by RNAi-mediated knock-down of either PI4KIIIbeta or PI4KIIalpha, but was greatly inhibited by knock-down of the PI4KIIIalpha enzyme. In HEK293 cells stably expressing the AT1a angiotensin receptor, agonist stimulation caused transient translocation of the PH-OSH2-tandem from the plasma membrane to the cytosol, indicating the PLC mediated hydrolysis of PI(4)P that paralleled the changes observed in 32P-labeled PI(4)P. The resynthesis of PI(4)P - whether monitored by the PH-OSH2-tandem translocation or by metabolic labeling - was inhibited by 10 ?M wortmannin or 10 ?M PAO, but not by a specific inhibitor of the PI4KIIIbeta enzyme. Similar inhibitor sensitivity was found when the AngII-induced InsP3 and the cytoplasmic Ca2+ signals were analyzed. These observations suggest that synthesis of the agonist-regulated PI(4)P and PI(4,5)P2 pools is carried out by the PI4KIIIalpha enzyme. Further studies are in progress to determine how this PI4K isofom that is primarily localized in the ER generates PI(4)P in the plasma membrane. Pleckstrin homology (PH) domains are small protein modules found in many signaling proteins that mediate the interaction of proteins with cellular membranes and also confer regulation by inositol phospholipids. Many PH domains are able to interact with specific phosphoinositides even when isolated from their parent proteins, and this feature has made them very popular to visualize phosphoinositides in living cells. Overexpression of these PH domains also interferes with cellular responses and we showed previously that distinct PH domains capable of binding the lipid, PI(3,4,5)P3 have selective inhibitory effects on certain PI(3,4,5)P3-mediated cellular responses, but not on others. This finding raised the possibility that the inhibitory effects of these PH domains is not solely due to their sequestration of the inositol lipid in question, but also due to sequestering protein binding partners resulting in more pathway-specific inhibition. To validate this assumption, we generated mutations on the surface of the PH domains of Akt and Grp1 that should not influence their lipid binding and membrane interaction, but could alter their protein-protein interaction. These studies yielded mutant PH domains that were indistinguishable from their wild-type counterparts in their inositol lipid or ?phosphate binding, but showed greatly reduced inhibition of Akt activation or of cell spreading (for AktPH and Grp1PH, respectively) when expressed in COS-7 cells. These data point to the importance of protein-protein interactions of PH domains in addition to their interaction with phosphoinositides. Further studies are in progress to determine the importance of these residues in the context of the whole molecule and to eventually identify the protein binding partner of the selected PH domains.